Bacterial Communities Associated with the Pine Wilt Disease Vector Monochamus Alternatus (Coleoptera: Cerambycidae) During Different Larval Instars
Total Page:16
File Type:pdf, Size:1020Kb
Journal of Insect Science, (2017)17(6): 115; 1–7 doi: 10.1093/jisesa/iex089 Research Article Bacterial Communities Associated With the Pine Wilt Disease Vector Monochamus alternatus (Coleoptera: Cerambycidae) During Different Larval Instars Xia Hu,1 Ming Li,1 Kenneth F. Raffa,2 Qiaoyu Luo,1 Huijing Fu,1 Songqing Wu,1 Guanghong Liang,1 Rong Wang,1 and Feiping Zhang1,3 1College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China, 2Department of Entomology, University of Wisconsin-Madison, 345 Russell Labs 1630 Linden Dr., Madison, WI 53706, and 3Corresponding author, e-mail: [email protected] Subject Editor: Campbell Mary and Lancette Josh Received 14 June 2017; Editorial decision 20 September 2017 Abstract We investigated the influence of larval instar on the structure of the gut bacterial community in the Japanese pine sawyer, Monochamus alternatus (Hope; Coleoptera: Cerambycidae). The diversity of the gut bacterial community in early, phloem-feeding larvae is significantly higher than in later, wood-feeding larvae. Many of these associates were assigned into a few taxonomic groups, of which Enterobacteriaceae was the most abundant order. The predominant bacterial genus varied during the five instars of larval development.Erwinia was the most abundant genus in the first and fifth instars,Enterobacter was predominant in the third and fourth instars, and the predominant genus in the second instars was in the Enterobacteriaceae (genus unclassified). Actinobacteria were reported in association with M. alternatus for the first time in this study. Cellulomonadaceae (Actinobacteria) was the second most abundant family in the first instar larvae (10.6%). These data contribute to our understanding of the relationships among gut bacteria and M. alternatus, and could aid the development of new pest control strategies. Key words: gut bacteria, pyrosequencing, long-horned beetle, Enterobacteriaceae Larvae of long-horned beetles (Cerambycidae) are xylophagous, and Iwasaki 1972, Teale et al. 2011). This insect-transmitted patho- which feed in subcortical tissues of healthy, dead, or decaying woody gen has caused significant losses of pines in Japan, Korea, China, and plants (Haack and Slansky 1987, Grünwald et al. 2010). Larval Portugal (Rodrigues 2009, Chen et al. 2013, Alves et al. 2016, Van development occurs entirely within the host, requires at least several Nguyen et al. 2017). One of the major strategies to manage the nem- months, and can kill trees (Allison et al. 2004). Bacterial communi- atode is to reduce between-tree transport by controlling M. alterna- ties associated with subcortically feeding beetles are known to play tus. Owing to the importance of M. alternatus, various aspects of its important roles in facilitating larvae in surviving and developing physiology and genetics have been studied, such as its pheromones within their host plants (Douglas 2009; Scully et al. 2013, 2014; (Teale et al. 2011), transcriptome (Wu et al. 2016), pathogens (Ma Alves et al. 2016). Bacterial communities are reported to contribute et al. 2009), symbiotic fungi (Maehara et al. 2005) and tracheal to their host beetles’ reproductive success, community interactions bacteria (Alves et al. 2016). However, symbiotic intestinal bacterial and niche diversification (Cardoza et al. 2006, Scott et al. 2008, communities are not well known for M. alternatus. Douglas 2009, Morales-Jiménez et al. 2013). Bacteria can contribute Like other Monochamus spp., M. alternatus feed on different to the nutrition of phloeophagous and xylophagous larvae, which sections of the wood during different larval stars. After hatching, rely on a nutrient-poor food source, by exploiting nitrogen and car- early larvae feed first on phloem under bark. Later larvae feed in the bon compounds in woody substrates and providing nutritional sup- xylem, including sapwood and heartwood, and form long, irregular plements that are absent from the substrate, such as amino acids mines (Yanega 1996). Thus, their food source changes substantially and essential vitamins (Dillon and Dillon 2004, Geib et al. 2008, during development, and previous studies have not yet categorized Morales-Jiménez et al. 2009, Berasategui et al. 2017). how corresponding gut communities respond (Park et al. 2007, Ma The Japanese pine sawyer, Monochamus alternatus (Hope; et al. 2009, Scully et al. 2014, Alves et al. 2016). Coleoptera: Cerambycidae), is the most important vector in Asia A deeper understanding of the structure of the microbiome of the for long-distance transport of the pine wood nematode (PWN), insect vector is required, and may contribute to the development of Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970, the new approaches to managing PWD. To better understand how do invasive pathogen that causes pine wilt disease (PWD), (Morimoto symbiotic intestinal bacterial communities relate to larval feeding © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ 1 licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2017, Vol. 17, No. 6 stage, we used a metagenomics approach to investigate the gut-asso- platform (San Diego, CA) for sequencing according to the stand- ciated bacteria diversity and community structures. ard protocols at Majorbio Bio-Pharm Technology, Shanghai, China. Raw fastq files were then demultiplexed and quality-filtered by using Materials and Methods QIIME (version 1.17). After pyrosequencing, the quality of the raw Miseq sequenc- Insect Collection and Dissection ing reads was checked with FastQC (Margulies et al. 2005, Andrews Larvae of M. alternatus in different instars were removed from 2014). Raw reads were quality screened by using an average minimum recently attacked Pinus massoniana (Lamb; Pinales: Pinaceae). quality score of 20. Barcodes and primers sequence were trimmed by Larvae in the first and second instar were collected from phloem and using the Trimmomatic. After quality control and barcode assignment, third, fourth, and fifth instar larvae were collected from sapwood operational taxonomic units (OTUs) were clustered with 97% simi- and heartwood. Larvae were placed on ice, and then transported to larity cutoff using Usearch (version 7.1) and chimeric sequences were the laboratory in sterile vials containing sterile moist paper. Sampling identified and removed using UCHIME. Mothur (http://www.mothur. was performed in the town of Guan Tou, Lianjiang county in Fujian org/) was used to sort sequences exactly matching the specific barcodes Province (N 26.15046°; E 119.59261°) in August 2015. All larvae into different samples. Then, Sickle tool (https://github.com/najoshi/ were manually removed directly from galleries using fine forceps. sickle) was used to perform the quality filtering to remove the reads with Instars first through fifth were separated according to the width their average quality score <50 or with any unknown bases. Then, the reads head capsules (Liu et al. 2008) (Table 1). Three larvae in each instar were assembled by Mothur command ‘make.contigs’ with the criteria of were prepared for 16S rRNA analysis, for a total of 15 samples. ‘maxambig = 0’, ‘maxhomop = 8’, and ‘minoverlap = 10’. The quality-fil- The larvae were surface sterilized with 70% ethanol for 1 min, tered reads were then processed by Mothur with commands ‘trim.seq’, and then rinsed twice with sterile water. After placing in 10 mM ster- ‘pre.cluster’, and ‘chimera.uchime’ to remove chimera and sequencing ilized phosphate-buffered saline (138 mM NaCl and 2.7 mM KCl, noise (Guo et al. 2015). Taxonomic classification of each sample was pH 7.4), the larvae were dissected under a stereomicroscope using individually conducted using Ribosomal Database Project (RDP) (http:// insect pins to obtain mid-guts and hindguts. One gut from each larva rdp.cme.msu.edu/) Classifier (version 2.6) with a confidence threshold was transferred to a 1.5-ml microcentrifuge tube with 500 ml of tris- of 50% (DeSantis et al. 2006, Wang et al. 2007, Cole et al. 2009, Quast EDTA (10 mM tris-HCl [pH 8.0], 1 mM EDTA) separately and then et al. 2013). ‘Aligner’ and ‘Complete Linkage Clustering’ were applied to homogenized several times with a plastic pestle, followed by vortex- calculate richness and diversity indices including OTUs, Shannon Index, ing for 3 min at the speed of 2500 r/min. The homogenate was cen- and Chaos index (Schloss et al. 2011). Sequences were rarefied to the trifuged at 4000 r/min for 15 s to separate the microbial cells from lowest number of reads in the samples using QIIME script single_rar- the gut wall tissues and undigested food (Hu et al. 2013). The super- efaction.py. before statistical analysis. Rarefaction curve methodology natant (containing bacteria) was transferred to new tubes for DNA was used to estimate the relationship between the expected OTU rich- extraction. All procedures were completed in a sterile environment. ness and sampling depth (Colwell et al. 2004). DNA Extraction and PCR Amplification Statistical Analysis DNA was extracted from the samples using QIAamp Fast DNA Principal component analysis (PCA) was performed using the vegan Stool Mini Kit (Qiagen,